Abstract

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.

DNA molecules, each containing a nucleosome and a bound Gal4DBD, were unzipped. All unzipped DNA molecules used in this work were in the region of 600 bp to 1.2 kbp. For clarity, much smaller regions are shown in all figures, with the origin of a template sequence defined as center position (the dyad) of the 601NPE. Shaded regions indicate locations of the Gal4 binding sequence and the 601NPE. (top panel) Cartoon illustrating the unzipping template design using for this experiment. A Gal4 sequence was separated from a 601NPE by 10 bp. The orientation of the 601NPE sequence is indicated by a white arrow. (middle panel) Unzipping in the direction in which the bound Gal4DBD was encountered first. (bottom panel) Unzipping in the direction in which the nucleosome was encountered first. DOI:http://dx.doi.org/10.7554/eLife.06249.003

Figure 1—figure supplement 1.. Unzipping experimental configuration.

Figure 1—figure supplement 1.. Unzipping experimental configuration.

8

The DNA template was attached, at one end,…

Figure 1—figure supplement 1.. Unzipping experimental configuration.

The DNA template was attached, at one end, to the surface of a glass coverslip via a digoxigenin–antidigoxigenin linkage, and at its other end to a microsphere via a biotin–streptavidin linkage. As the coverslip was moved away from the trapped microsphere, using a loading-rate clamp, the dsDNA was sequentially converted into ssDNA upon base pair separation. The presence of force peaks above the naked DNA baseline revealed the detected locations of protein–DNA interactions. DOI:http://dx.doi.org/10.7554/eLife.06249.004

Figure 1—figure supplement 2.. Characterization of the…

Figure 1—figure supplement 2.. Characterization of the precision and accuracy of detection of the locations…

Figure 1—figure supplement 2.. Characterization of the precision and accuracy of detection of the locations of Gal4DBD and nucleosome.

Single molecule unzipping detected Gal4DBD and a nucleosome simultaneously. The histograms for detected locations of Gal4DBD (green) and nucleosome (red) were obtained by pooling data from multiple single molecule traces, with the expected bound locations represented by their respective dashed lines. For each histogram, the precision was determined by the standard deviation of each histogram, and the accuracy by the difference between the mean of the histogram and the expected value (the vertical dashed line). These data demonstrate both the precision and accuracy to be near base-pair. DOI:http://dx.doi.org/10.7554/eLife.06249.005

Figure 1—figure supplement 3.. Characterization of Gal4DBD…

Figure 1—figure supplement 3.. Characterization of Gal4DBD binding.

7

To characterize Gal4DBD binding to its binding…

Figure 1—figure supplement 3.. Characterization of Gal4DBD binding.

To characterize Gal4DBD binding to its binding sequence, DNA unzipping was carried out in the presence of known Gal4DBD concentrations. (A) A representative unzipping trace of a bound Gal4DBD. The location of the binding sequence is shown as a shaded region. Naked DNA unzipping baseline is shown in gray. (B) Fraction of bound Gal4DBD vs the concentration of Gal4DBD. For a given concentration of Gal4DBD, measurements were on multiple DNA molecules to obtain the fraction of Gal4DBD. Data points are represented as (mean ± s.e.m.). The relation for the fraction bound vs [Gal4DBD] was fit to:[Gal4DBD][Gal4DBD]+Kd (red smooth curve), which yielded the dissociation equilibrium constant Kd= 3.4 nM. (C) Fraction of bound Gal4DBD vs time. This relation shows no significant Gal4DBD dissociation from its binding sequence over a course of one hour. Data were fit to a straight line to guide the eye. DOI:http://dx.doi.org/10.7554/eLife.06249.006

Figure 1—figure supplement 4.. Detection of Gal4DBD…

Figure 1—figure supplement 4.. Detection of Gal4DBD binding.

7

The presence of a bound Gal4DBD was…

Figure 1—figure supplement 4.. Detection of Gal4DBD binding.

The presence of a bound Gal4DBD was determined by the magnitude of the force peak at the Gal4 binding sequence. In the presence of a bound Gal4DBD, the peak force increased substantially and was readily differentiable from the baseline DNA force. DOI:http://dx.doi.org/10.7554/eLife.06249.007

Nucleosomes were remodeled by either 1 nM ISW1a or 1 nM SWI/SNF with 1 mM ATP for 1 min, a time sufficiently short that the majority of nucleosomes were not remodeled (Figure 2—figure supplement 2). Each DNA template was subsequently unzipped. For templates used in (D)–(G), the 601NPE was separated from the Gal4 binding sequence by 10 bp. (A) Distribution of the location of a nucleosome before remodeling. Data were pooled from measurements on multiple nucleosomal DNA molecules. (B) Distribution of the location of a nucleosome remodeled by ISW1a in the absence of Gal4DBD. (C) Distribution of the location of a nucleosome remodeled by SWI/SNF in the absence of Gal4DBD. (D) Distribution of the location of a nucleosome remodeled by ISW1a with a bound Gal4DBD initially located upstream of the 601NPE. (E) Distribution of the location of a nucleosome remodeled by SWI/SNF with a bound Gal4DBD initially located upstream of the 601NPE. (F) Distribution of the location of the nucleosome remodeled by ISW1a with a bound Gal4DBD initially located downstream of the 601NPE. (G) Distribution of the location of a nucleosome remodeled by SWI/SNF with a bound Gal4DBD initially located downstream of the 601NPE. DOI:http://dx.doi.org/10.7554/eLife.06249.008

Figure 2—figure supplement 1.. Directionality of ISW1a…

Figure 2—figure supplement 1.. Directionality of ISW1a and SWI/SNF remodeling of a nucleosome in close…

Figure 2—figure supplement 1.. Directionality of ISW1a and SWI/SNF remodeling of a nucleosome in close proximity to a bound Gal4DBD.

The figure panels A to G show the raw traces for the corresponding panels in Figure 2. DOI:http://dx.doi.org/10.7554/eLife.06249.010

(A) For each nucleosome distribution, remodeled by either ISW1a or SWI/SNF as shown in Figure 2B,C, was fit to a double Gaussian function. One Gaussian (narrow green curve) corresponds to the distribution of unremodeled nucleosomes and the other (broader green curve) to the distribution of remodeled nucleosomes. The sum of the two Gaussians is shown as the red curve. (B) A table summarizing fractions of unremodeled percentage and remodeled percentage for both ISW1a and SWI/SNF. DOI:http://dx.doi.org/10.7554/eLife.06249.011

Figure 3.. ISW1a remodeling is blocked by…

Figure 3.. ISW1a remodeling is blocked by a bound Gal4DBD.

5

Nucleosomes were remodeled by 1…

Figure 3.. ISW1a remodeling is blocked by a bound Gal4DBD.

Nucleosomes were remodeled by 1 nM ISW1a with 1 mM ATP for 10 min with or without Gal4DBD. Shaded regions indicate locations of Gal4 binding sequence and 601NPE. (A) Distributions of the locations of the nucleosome and bound Gal4DBD, either before remodeling or without Gal4DBD, as controls. (B) Distributions of the locations of the nucleosome after ISW1a remodeling in the presence of Gal4DBD on three different templates of increasing separation between the Gal4 binding sequence and the 601NPE. For each template, the nucleosome position distribution is dominated by a narrow population, but has a few outliers which were moved a much greater distance and some of which even passed the Gal4 binding sequence. These outliers (∼5%) were likely a result of templates that did not have a bound Gal4DBD initially (∼5%; see main text). This is further supported by the observation that none of these outlier traces revealed a bound Gal4DBD. Nonetheless, in order to avoid possible bias, these nucleosome positions were still used for further analysis in (C) and thus contributed to the error bars in (C). (C) Relationship between the distance the remodeled nucleosome moved and the separation between the Gal4 binding sequence and the 601NPE. Error bars are SEM. DOI:http://dx.doi.org/10.7554/eLife.06249.012

Shown is a representative unzipping trace of a DNA molecule containing an end-positioned nucleosome and a bound Gal4DBD. DOI:http://dx.doi.org/10.7554/eLife.06249.013

Figure 3—figure supplement 2.. ISW1a centers a…

Figure 3—figure supplement 2.. ISW1a centers a nucleosome between a bound Gal4DBD and the template…

Figure 3—figure supplement 2.. ISW1a centers a nucleosome between a bound Gal4DBD and the template end.

This relation shows that ISW1a remodeler tends to center a nucleosome between the Gal4 binding site and the DNA end. Error bars are SEM. The dashed line indicates the center position on the DNA. DOI:http://dx.doi.org/10.7554/eLife.06249.014

Nucleosomes were remodeled by 1.5 nM SWI/SNF with 1 mM ATP for 10 min with or without Gal4DBD. Shaded regions indicate locations of Gal4 binding sequence and 601NPE. (A) Distributions of the locations of the nucleosome and bound Gal4DBD before remodeling (upper plot), after remodeling without Gal4DBD (middle plot), and after remodeling with Gal4DBD (lower plot). (B) Representative traces in the case of before remodeling (top plot; N = 55) and after remodeling (middle and bottom plots; N = 50). The middle plot shows an example trace where a nucleosome was remodeled to the opposite side of Gal4DBD relative to its original position; while the bottom plot shows an example trace where a nucleosome was remodeled to the same side of Gal4DBD relative to its original position. Gray traces were taken from the corresponding naked DNA. DOI:http://dx.doi.org/10.7554/eLife.06249.015

Figure 4—figure supplement 1.. Distributions of the locations of SWI/SNF remodeled nucleosomes as determined by unzipping from both directions.

To determine whether the distributions of the locations of remodeled nucleosomes were similar for measurements made by unzipping the DNA in one direction vs in the other direction, we unzipped multiple DNA molecules, each containing either an unremodeled or a remodeled nucleosome, from both directions. Nucleosome remodeling was carried out in 1 nM SWI/SNF with 1 mM ATP for 5 min. Our data show similar distributions for data obtained in both directions. DOI:http://dx.doi.org/10.7554/eLife.06249.017

Figure 4—figure supplement 2.. Nucleosome remodeling by…

Figure 4—figure supplement 2.. Nucleosome remodeling by SWI/SNF on a template with the Gal4 binding…

Figure 4—figure supplement 2.. Nucleosome remodeling by SWI/SNF on a template with the Gal4 binding site separated from the 601NPE by 24 bp.

To test whether SWI/SNF is able to evict Gal4DBD via nucleosome remodeling when a bound Gal4DBD is located farther away from a nucleosome, we used a template where the Gal4 binding site was separated from the 601NPS by 24 bp and carried out unzipping experiments under identical conditions as those shown in Figure 4. Out of all traces where the nucleosome was repositioned to the opposite side of the Gal4 binding site by SWI/SNF (N = 13), we did not detect any Gal4DBD binding signature on the template, indicating eviction of Gal4DBD. Shown are example traces, with arrows indicating the unzipping directions. DOI:http://dx.doi.org/10.7554/eLife.06249.018

Shaded regions indicate locations of Lac repressor binding sequence and 601NPE. (A) Distributions of locations of nucleosomes before remodeling (upper plot), after remodeling by ISW1a without Lac repressor (middle plot), and after remodeling by ISW1a with Lac repressor (lower plot). Lac repressor binding sequence was separated from the 601NPE by 50 bp. Nucleosomes remodeling was carried out in 1 nM ISW1a with 1 mM ATP for 10 min with or without Lac repressor. (B) Representative traces in the case of before SWI/SNF remodeling (top plot; N = 25) and after remodeling (middle and bottom plots; N = 27). Lac repressor binding site was separated from the 601NPE by 10 bp. Nucleosomes were remodeled by 1.5 nM SWI/SNF with 1 mM ATP for 10 min. The middle plot shows an example trace where a nucleosome was remodeled to the other side of the Lac repressor and the bottom plot shows an example trace where a nucleosome was remodeled to the same side of Lac repressor. DOI:http://dx.doi.org/10.7554/eLife.06249.019

Figure 6.. The interplay between nucleosome remodeling…

Figure 6.. The interplay between nucleosome remodeling and a bound TF.

4

When a nucleosome is…

Figure 6.. The interplay between nucleosome remodeling and a bound TF.

When a nucleosome is remodeled by ISW1a (left), nucleosome repositioning is blocked by a bound TF, the TF remains intact, and the remodeled nucleosome is positioned with the TF acting as a boundary and reference point. On the other hand, when a nucleosome is remodeled by SWI/SNF (right), nucleosome positioning is unaffected by a bound TF and the TF is evicted. DOI:http://dx.doi.org/10.7554/eLife.06249.020